Effects of glutamine deprivation on glutamine transport and synthesis in primary culture of rat skeletal muscle.

The effects of deprivation and supplementation of exogenous glutamine (0.06 and 2.2 mM in the culture medium, respectively) were studied in mononucleated myoblasts and in multinucleated myotubes. Myoblasts cultured in glutamine-deprived medium showed reductions in plating efficiency and myotube fusion index. Myotubes grown in glutamine-supplemented cultures had higher intracellular glutamine concentrations than those grown in glutamine-deprived medium (67 +/- 4.2 vs. 46 +/- 3.6 nmol/mg cell protein, respectively) and glutamine-supplemented myotubes utilized glutamine, whereas glutamine-deprived myotubes released it. Glutamine deprivation for 12 h caused a significant, cycloheximide-blockable increase in the capacity for glutamine uptake via system Nm in both myoblasts and myotubes (maximum velocity increases of 23 +/- 5.3 and 35 +/- 4.2%, respectively), which was reversed by glutamine replenishment. Depriving myotubes of glutamine did not alter the kinetics of uptake of amino acid transport systems A, ASC, or L. Glutamine deprivation resulted in a threefold increase in glutamine synthetase activity, whereas glutaminase activity remained unchanged. System Nm and glutamine synthetase appear to undergo adaptive upregulation in glutamine-deprived muscle cells to compensate for the reduced exogenous glutamine supply.

A role for membrane transport in modulation of intramuscular free glutamine turnover in streptozotocin diabetic rats.

We wished to examine the effects of diabetes on muscle glutamine kinetics. Accordingly, female Wistar rats (200 g) were made diabetic by a single injection of streptozotocin (85 mg/kg) and studied 4 days later; control rats received saline. In diabetic rats, glutamine concentration of gastrocnemius muscle was 33% less than in control rats: 2.60 +/- 0.06 mumol/g vs. 3.84 +/- 0.13 mumol/g (P < 0.001). In gastrocnemius muscle, glutamine synthetase activity (Vmax) was unaltered by diabetes (approx. 235 nmol/min per g) but glutaminase Vmax increased from 146 +/- 29 to 401 +/- 94 nmol/min per g; substrate Km values of neither enzyme were affected by diabetes. Net glutamine efflux (A-V concentration difference x blood flow) from hindlimbs of diabetic rats in vivo was greater than control values (-30.0 +/- 3.2 vs. -1.9 +/- 2.6 nmol/min per g (P < 0.001)) and hindlimb NH3 uptake was concomitantly greater (about 27 nmol/min per g). The glutamine transport capacity (Vmax) of the Na-dependent System Nm in perfused hindlimb muscle was 29% lower in diabetic rats than in controls (820 +/- 50 vs. 1160 +/- 80 nmol/min per g (P < 0.01)), but transporter Km was the same in both groups (9.2 +/- 0.5 mM). The difference between inward and net glutamine fluxes indicated that glutamine efflux in perfused hindlimbs was stimulated in diabetes at physiological perfusate glutamine (0.5 mM); ammonia (1 mM in perfusate) had little effect on net glutamine flux in control and diabetic muscles. Intramuscular Na+ was 26% greater in diabetic (13.2 mumol/g) than control muscle, but muscle K+ (100 mumol/g) was similar. The accelerated rate of glutamine release from skeletal muscle and the lower muscle free glutamine concentration observed in diabetes may result from a combination of: (i), a diminished Na+ electrochemical gradient (i.e., the net driving force for glutamine accrual in muscle falls); (ii), a faster turnover of glutamine in muscle and (iii), an increased Vmax/Km for sarcolemmal glutamine efflux.